Fluorescence detection enhancement using photonic crystal extraction

ABSTRACT

Enhancement of fluorescence emission from fluorophores bound to a sample and present on the surface of two-dimensional photonic crystals is described. The enhancement of fluorescence is achieved by the combination of high intensity near-fields and strong coherent scattering effects, attributed to leaky photonic crystal eigenmodes (resonance modes). The photonic crystal simultaneously exhibits resonance modes which overlap both the absorption and emission wavelengths of the fluorophore. A significant enhancement in fluorescence intensity from the fluorophores on the photonic crystal surface is demonstrated.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. application Ser. No. 11/986,156 filedNov. 19, 2007, which claims priority under 35 U.S.C. §119(e) to U.S.Provisional Application Ser. No. 60/916,462 filed May 7, 2007, thecontent of which is incorporated by reference herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made, at least in part, with United Statesgovernmental support awarded by the National Science Foundation underNSF BES 0427657. The United States Government has certain rights in thisinvention.

BACKGROUND OF THE INVENTION

Photonic crystals, also commonly referred to as photonic bandgapstructures, are periodic dielectric structures exhibiting a spatiallyperiodic variation in refractive index that forbids propagation ofcertain frequencies of incident electromagnetic radiation. The photonicband gap of a photonic crystal refers to the range of frequencies ofelectromagnetic radiation for which propagation through the structure isprevented in particular directions. A photonic crystal structure may bedesigned to exhibit extraordinarily high reflection efficiency atparticular wavelengths, at which optical standing waves develop andresonate within the photonic crystal structure. Such optical resonancesare known to occur at the wavelengths adjacent to the photonic band gap,sometimes referred to as the photonic band edge. The spatial arrangementand refractive indices of these structural domains generate photonicbands gaps that inhibit propagation of electromagnetic radiationcentered about a particular frequency.

This anomalous resonant phenomenon (termed guided-mode resonance) arisesdue to the introduced periodicity which allows phase-matching ofexternally incident radiation into modes that can be reradiated intofree-space. Due to the fact that these modes possess finite lifetimeswithin such structures, they are referred to as ‘leaky eigenmodes’ ofthe structures. More recently, guided-mode resonances have been studiedin crossed gratings or two-dimensional (2D) photonic crystal (PC) slabs.The leaky nature of these modes has been exploited towards thedevelopment of light emitting diodes (LEDs) with improved extractionefficiency, biosensors (see Cunningham et al., Colorimetric ResonantReflection as a Direct Biochemical Assay Technique, Sensors andActuators B, 2002, 81, pgs 316-328 (2002)) and vertically emittinglasers.

The ability of photonic crystals to provide high quality factor (Q)resonant light coupling, high electromagnetic energy density, and tightoptical confinement can also be exploited to produce highly sensitivebiochemical sensors. Here, Q is a measure of the sharpness of the peakwavelength at the resonant frequency. Photonic crystal biosensors aredesigned to allow a liquid test sample to penetrate the periodiclattice, and to tune the resonant optical coupling condition throughmodification of the surface dielectric constant of the crystal throughthe attachment of biomolecules or cells. Due to the high Q of theresonance, and the strong interaction of coupled electromagnetic fieldswith surface-bound materials, several of the highest sensitivitybiosensor devices reported are derived from photonic crystals. Suchdevices have demonstrated the capability for detecting molecules withmolecular weights less than 200 Daltons (Da) with high signal to noisemargins, and for detecting individual cells. Because resonantly coupledlight within a photonic crystal can be effectively spatially confined, aphotonic crystal surface is capable of supporting large numbers ofsimultaneous biochemical assays in an array format, where neighboringregions within ˜10 μm of each other can be measured independently. SeeLi, P., B. Lin, J. Gerstenmaier, and B. T. Cunningham, A new method forlabel-free imaging of biomolecular interactions. Sensors and ActuatorsB, 2003.

Given substantial advances in their fabrication and their unique opticalproperties, photonic crystal-based sensors are under development for avariety of applications. Biosensors are one application. Biosensorsincorporating photonic crystal structures are described in the followingreferences, which are hereby incorporated by reference in theirentireties: U.S. Pat. Nos. 7,118,710, 7,094,595, and 6,990,259; U.S.Published applications 2007/0009968; 2002/0127565; 2003/0059855;2007/0009380; 2003/0027327; Cunningham, B. T. J. Qiu, P. Li, J. Pepperand B. Hugh, A Plastic calorimetric Resonant Optical Biosensor forMulti-parallel Detection of Label Free Biochemical Interactions, Sensorsand Actuators B, 2002, 85, pgs 219-226.

U.S. Pat. No. 6,707,561 describes a grating-based biosensing technologythat is sometimes referred to in the art as Evanescent Resonance (ER)technology. This technology employs a submicron scale grating structureto amplify a fluorescence signal, following a binding event on thegrating surface, where one of the bound molecules carries a fluorescentlabel. ER technology enhances the sensitivity of fluorophore basedassays enabling binding detection at analyte concentrationssignificantly lower than non-amplified assays.

ER technology uses grating generated optical resonance to concentratelaser light on the grating surface where binding has taken place. Inpractice, a laser scanner sweeps the sensor at some angle of incidence(θ), typically from above the grating, while a detector detectsfluoresced light (generally at longer optical wavelength) from thesensor surface. By design, ER grating optical properties result innearly 100% reflection, also known as resonance, at a specific angle ofincidence and laser wavelength (λ). Confinement of the laser light byand within the grating structure amplifies emission from fluorophoresbound within range of the evanescent field (typically 1-2 μm). Hence, atresonance, transmitted light intensity drops to near zero.

The spectral width and wavelength of the resonance phenomena describesthe important externally measurable parameters of a device. Resonancewidth refers to the full width at half maximum, in wavelength measure,of a resonance feature plotted as reflectance (or transmittance) versuswavelength. Resonance width also refers to the width, in degrees, of aresonance feature plotted on a curve representing reflectance ortransmittance as a function of θ. In practice, one can make adjustmentsto the incident angle to “tune” the resonance towards maximum laserfluorophore coupling.

In one embodiment of this invention, a biosensor is constructed as aphotonic crystal structure which has a periodic surface grating in whicha so-called evanescent resonance is created. Conceptually, resonancephenomena can occur in planar dielectric layer gratings where almost100% switching of optical energy between reflected and transmitted wavesoccurs when the grooves of the grating have sufficient depth and theradiation incident on the corrugated structure is at a particular angle.This phenomenon is exploited in the sensing area of the platform wherethat sensing area includes grating structures (e.g., grooves, or holesor posts) of sufficient depth and light is caused to be incident on thesensing area of the platform at an angle such that evanescent resonanceoccurs in that sensing region. This creates in the sensing region anenhanced evanescent field which is used to excite samples underinvestigation. It should be noted that the 100% switching referred toabove occurs with parallel beam and linearly polarized coherent lightand the effect of an enhanced evanescent field can also be achieved withnon-polarized light of a non-parallel focused laser beam. Excitationphotons incident on the sample (chip, for example) under resonanceconditions couple into a thin corrugated surface (such as a metal oxidelayer) at the site of incidence. As a result of the transducer geometry,the energy is locally confined into the thin corrugated layer of highrefractive index material. Consequently, strong electromagnetic fieldsare generated at the surface of the chip. The effect has been attributedas evanescent resonance and leads to increased fluorescence intensity ofchromophores (fluorescent material) close to the surface of the sensor.The effective field strength can be increased up to 100-fold by theconfinement of the available excitation energy, depending on the opticalproperties of the optical detection system used.

The inventive sensors and method of this disclosure are useful inconjunction with a variety of different types of fluorophores. Suchfluorophores have excitation and emission spectra which are typicallywell characterized and available from the manufacturer, or can bedetermined experimentally.

Quantum Dots (QDs) are fluorescent, nanometer-sized inorganicsemiconductor crystals that have rapidly emerged as an important classof nanomaterials which promise to revolutionize a wide range ofnanotechnology-enabled fields. QDs derive their unique opticalproperties (broad absorption spectrum, narrow, size-tunable emissionspectrum, high photostability, quantum efficiency and strong nonlinearresponse) from quantum confinement effects. These attributes, coupledwith the ability to functionalize QDs, has made them importantcandidates for light sources, solar cells, optical switches andfluorescent probes in sensitive biological assays. The ability to moreefficiently excite and extract the light emitted by QDs would thus be ofvital importance in realizing high brightness light sources, enhancednonlinear effects and lowering the detection limits in biologicalassays.

Fluorescent dyes represent a broad class of organic and inorganicfluorescent molecules that are capable of emitting light. Generally,electrons within the fluorescent molecule are excited from a groundstate to an excited state through the absorption of a photon from anexternal source of illumination. The electron in the excited state mayreturn to the ground state through a variety of mechanisms. One suchmechanism is through the release of heat in the form of a phonon.Another such mechanism is through the release of light in the form of aphoton. Absorption of energy by the fluorophore occurs at a particularrange of incident photon energies (or equivalently wavelengths) that areunique for each type of molecule. Due to conservation of energy, theemitted photon energy must be less than or equal to the energy of theincident photon, and therefore the emitted wavelength must be largerthan the incident photon wavelength. Therefore, a fluorescent moleculehas two distinct spectra associated with it: the range of wavelengthsfor which it is capable of absorbing photons, and the range ofwavelengths for which it is capable of emitting photons. The differencebetween the absorption and emission wavelength is known as the Stokesshift.

SUMMARY

Photonic crystal sensors are disclosed for use in testing samples inwhich a fluorophore, e.g., inorganic crystalline semiconductor (“quantumdot”) or fluorescent dye is present in the sample. The sample andfluorescent dye are in close proximity, or more typically bound, to thephotonic crystal surface, e.g., by depositing the sample withfluorophore on the sensor surface in a dry or aqueous environment.

In one aspect of this disclosure, the photonic crystal sensor isconstructed and arranged with a surface in the form of a periodicsurface grating structure which simultaneously exhibits multipleresonance modes for light at a given incident angle. The resonance modesoverlap both the excitation and emission spectra of the fluorophore. Inparticular, when light is incident upon the photonic crystal at anappropriate incident angle θ, the photonic crystal sensor simultaneouslyexhibits multiple resonance modes (referred to herein as leakingeigenmodes or leaky modes) which have spectra that overlap both theabsorption (excitation) and emission spectra of the fluorophore presentin the sample. In this document, the term “spectrum” in the context of aresonant mode refers to the band of wavelengths of incident light inwhich a guided mode resonance is created in the photonic crystal as theangle of incidence θ varies. A photonic crystal constructed and arrangedso as to possess such a doubly resonant scheme (i.e., exhibitingresonance modes overlapping both the excitation and emission spectra ofthe fluorophore simultaneously at a given incident angle θ) yieldsstrongly enhanced fluorescent emission and the ability to extract suchemission in a highly efficient manner, resulting in a high sensitivesensors suitable for a very broad range of applications, as will beexplained below.

A sample testing system for testing a sample having a fluorophore boundto the sample is also described. The testing system includes a detectioninstrument comprising a light source and a detector; and a photoniccrystal sensor having a periodic grating structure. The sample includingthe fluorophore is placed on the periodic grating structure. The lightsource of the detection system is oriented relative to the photoniccrystal sensor such that the light source illuminates the photoniccrystal sensor at a incident angle θ in which the photonic crystalsimultaneously exhibits a plurality of resonant modes, the resonantmodes including an excitation mode having a first resonant spectrum andan extraction mode having a second resonant spectrum. The periodicgrating structure is constructed and arranged such that the resonantspectrum of the photonic crystal in the excitation mode at leastpartially overlaps the excitation spectrum of the fluorophore and theresonant spectrum in the extraction mode at least partially overlaps theemission spectrum of the fluorophore. The detector operates to detectradiation from the fluorophore in the emission spectrum.

A method of testing a sample with a fluorophore present in the samplewith the photonic crystal sensors of this disclosure are also described.The method includes the step of placing the sample onto the surface of aphotonic crystal sensor; illuminating the photonic crystal biosensorwith light at an angle of incidence θ, the biosensor responsively andsimultaneously exhibiting (1) an excitation resonance mode having aspectrum which at least partially overlaps the excitation spectrum ofthe fluorophore; and (2) an extraction resonance mode having a spectrumwhich at least partially overlaps the emission spectrum of thefluorophore, the illumination and the resulting excitation andextraction resonance modes causing the fluorophore to emit light; andcollecting the emitted light from the fluorophore and directing theemitted light onto a detector.

In yet another aspect, a photonic crystal sensor is disclosed whichincludes a periodic surface grating structure which exhibits a resonancemode at a given incident angle which overlaps the emission spectrum of afluorophore which is present with a sample deposited on the sensor, anddoes not have a resonance mode which overlaps the excitation spectrum ofthe fluorophore. The photonic crystal sensor produces an enhancedextraction effect without also producing an enhanced excitation effect.Sample testing systems suitable for the doubly resonant photonic crystalsensors are also useful with this embodiment.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is an illustration of a photonic crystal featuring the enhancedexcitation and extraction features of this disclosure and comparing thedirectionally enhanced extraction of emitted radiation from fluorophoresfrom the photonic crystal with a dielectric slab which does not featurethe resonance modes of this disclosure.

FIG. 2 a is an illustration of a two-dimensional photonic crystal devicehaving a periodic surface grating structure constructed as a twodimensional array of holes in accordance with one exemplary embodimentof the invention. FIG. 2 b shows Scanning Electron Micrograph (SEM)images of a cleaved photonic crystal device of FIG. 2 a. In FIG. 2 a,the lines joining the features Γ, X and M are axes of high symmetry inthe photonic crystal surface. Λ=300 nm is the period of the surfacegrating structure (with a square unit cell in X and Y directions) andt=125 nm, the thickness of a high index of refraction material layerwhich is deposited onto the grating layer. Θ is the angle the incidentbeam of light makes with the vertical.

FIGS. 3 a and 3 b are graphs of the calculated and experimentaldispersion spectra, respectively, of the fabricated photonic-crystalsensor of FIGS. 2 a and 2 b for white, S-polarized incident illuminationincident along the Γ-M direction. The sensor exhibits resonant mode atλ=488 nm when the incident beam makes an angle θ=11.2° with the surfacenormal. The excitation of the resonant mode at this wavelength, wherethe quantum dot fluorophore is strongly absorbing, provides the requirednear-field enhancement for evanescent resonance (enhanced excitation).The shading scale shows the efficiency of transmission. Higher Q factorsfor a resonance mode are indicated by thinner lines whereas broader Qfactors for a resonance mode are indicated by relatively thicker lines.

FIGS. 4 a and 4 b are calculated near electric-field intensities (E²) atresonance for the photonic crystal structure of FIG. 2 a, with FIG. 4 ashowing the intensity for the lower surface of the surface grating(bottom of the hole) and FIG. 4 b showing the intensity for the uppersurface of the surface grating. The intensities are for the leaky mode(resonance) with λ=488 nm when the incident beam makes an angle θ=11.2°with the surface normal. The intensity is normalized to the unitamplitude incident wave.

FIGS. 5 a, 5 b and 5 c are graphs of the dispersion spectrum showing theresonance modes of the photonic crystal sensor showing the possibilityof enhanced extraction for all polarizations and directions of incidentwhite light. The graphs were experimentally determined from the photoniccrystal of FIG. 2 a. FIG. 5 a is the graph for P-polarized and incidentalong the Γ-M direction, FIG. 5 b is the graph for P-polarized andincident along the Γ-X direction, and FIG. 5 c is the graph forS-polarized, incident along Γ-X direction.

FIGS. 6 a and 6 b are fluorescence (pseudocolor) scan images of thephotonic crystal of FIG. 2 a with quantum dots dispensed on the surface.FIG. 6 a is a scan image taken when the photonic crystal is resonantwith respect to the incident beam (θ=11.2°), showing an enhancementfactor of over 108 times. FIG. 6 b is a scan image taken when thephotonic crystal is not resonant with the incident beam (θ=0°), showingan enhancement factor of over 13 times. The circular regions representthe area over which intensity information was averaged. In both theimages, the circle to the left shows the control region where nophotonic crystal is present.

FIG. 7 is a near-field scanning optical microscopy (NSOM) image of thenear-fields on the surface of a fabricated photonic crystal device.

FIG. 8 is an illustration of an angle-resolved fluorescence measurementfrom the photonic crystal surface for the Γ-X direction.

FIG. 9 is a graph of the normalized fluorescence spectrum of a quantumdot fluorescence as modified by the photonic crystal sensor.

FIG. 10 illustrates angle-resolved fluorescence measurements from thephotonic crystal surface for the light polarized in the Γ-M direction.

FIG. 11 is a graph of the intensity of quantum dot emission as afunction of time.

FIG. 12 is a block diagram of a detection instrument for a photoniccrystal sensor featuring enhanced excitation and enhanced extraction.

DETAILED DESCRIPTION

Photonic crystal sensors are disclosed for use in testing samples inwhich a fluorophore, e.g., inorganic crystalline semiconductor (“quantumdot”) or fluorescent dye is present in the sample. The sample andfluorescent dye are in close proximity, or more typically bound, to thephotonic crystal surface, e.g., by depositing the sample withfluorophore on the sensor surface in a dry or aqueous environment. Anexample of the photonic crystal is shown in FIG. 2 a and will bedescribed in detail subsequently.

In one aspect of this disclosure, the photonic crystal sensor isconstructed and arranged with a surface in the form of a periodicsurface grating structure (such as a two dimensional array of holesshown in FIG. 2 a), which simultaneously exhibits multiple resonancemodes for light at a given incident angle. The resonance modes are showngraphically in FIGS. 4 and 6 (transmission efficiency shown plotted as afunction of wavelength for incident light at different angles tovertical), described subsequently, and are indicated in the areas of thegraphs where the transmission efficiency drops to zero. The resonancemodes overlap both the excitation and emission spectra of thefluorophore present in the sample. In particular, when light is incidentupon the photonic crystal at an appropriate incident angle θ, thephotonic crystal sensor simultaneously exhibits multiple resonance modes(referred to herein as leaking eigenmodes or leaky modes) which havespectra that overlap both the absorption (excitation) and emissionspectra of the fluorophore present in the sample. A photonic crystalconstructed and arranged so as to possess such a doubly resonant scheme(i.e., exhibiting resonance modes overlapping both the excitation andemission spectra of the fluorophore) to enhance fluorescent emissionfrom the fluorophore has not, to the current knowledge of the inventors,been previously demonstrated.

The presence of the photonic crystal resonance peak occurring at thefluorescence excitation wavelength gives rise to the formation of highintensity electromagnetic near-fields which serves to efficiently excitethe fluorophore present in the sample. This phenomenon is referred toherein interchangeably as “evanescent resonance” or “enhancedfluorescence.”

The additional feature of a resonance mode in the photonic crystal whichoverlaps the emission spectrum of the fluorophore serves as an effectivemechanism to extract this enhanced emission. In particular, a photoniccrystal with resonance modes overlapping both the emission andexcitation spectra of the fluorophore increases the number offluorescence emitted photons which can be gathered by a detector in anassociated measuring instrument for use with the photonic crystalsensor. Photonic crystal resonance occurring at the emission wavelengthof the fluorophore can be used to efficiently couple emitted photons atthe photonic crystal surface to be selectively directed into free spaceat a particular exit angle, instead of uniformly directed in alldirections. This phenomenon of selective direction of emitted photons isbelieved to be due to fluorescence coupling to the overlapping leakymodes producing Bragg scattering out of the structure, thereby greatlyreducing the amount of light trapped in the photonic crystal sensor inthe extraction mode. If the dispersion of these overlapping emissionleaky modes is close to the Γ-point band edge (i.e., the magnitude ofthe in-plane wave vector for incident polarized light is close to zero),a significant amount of the emitted light can be extracted from thephotonic crystal sensor within small angles of the vertical. Thisdiscovery allows for positioning of a detector (or associated opticalelements such as fiber optic probe which is coupled to a detector) atthe correct position relative to the photonic crystal surface, andallows for capturing more photons that would otherwise occur, e.g., ascompared to a fluorophore emitting from a non-photonic crystal surface.

A photonic crystal sensor as just described is shown in FIG. 1 as item10, with a dielectric slab 20 shown next to it which does not possess aphotonic crystal property for purposes of comparison. The photoniccrystal sensor 10 consists of a periodic surface grating structureformed on its surface 11, which in this example takes the form of atwo-dimensional array of holes 12. (Other periodic structures for thephotonic crystal 10 are possible, as will be explained below). A samplecontaining a fluorophore 14 is applied to the surface 11. The photoniccrystal is illuminated with light (in this case from above) at an angleθ relative to the vertical direction. The angle θ is shown in FIG. 2 a.The properties of the periodic grating (holes) 14 on the surface 11 aresuch that the photonic crystal simultaneously exhibits resonance modeswhich overlap both the excitation and emission spectra of thefluorophore 14. The overlap of the resonance mode with the excitationspectrum produces an enhanced excitation of the fluorophore 14. Theoverlap of the resonance mode with the emission spectrum of thefluorophore 14 produces a directionally enhanced extraction of theemitted radiation due to Bragg scattering, which is indicated by thelines 16 all pointing in the same direction. As a practical matter, theangle of the lines 16 can be determined either by simulation or byexperimentation and light collection apparatus placed in alignment withthe lines 16 so as to collect this radiation. The light collectionapparatus (e.g., fiber optic probe) supplies the collected radiation toa detection device such as a CCD imager or photomultiplier tube so as tomake measurements or collect images of the radiation, thereby obtaininginformation as to the sample.

Consider now the dielectric slab 20 in FIG. 1 which does not possess anyphotonic crystal attributes for the sake of comparison. Assuming theincident light encompasses the excitation spectrum of the fluorophore14, at least some fluorescence can be expected. However, because thereis no resonant mode in the slab 20 overlapping the emission spectrum ofthe fluorophore, the resulting fluorescence is radiated in an almostspatially uniform manner, indicated by the lines 16 pointing in alldirections. Thus, the concentration of radiation in the same directionas indicated at 16 on the left hand side of FIG. 1 produces an enhancedextraction of the fluorescent signal which is not present in anon-photonic crystal structure such as shown on the right hand side ofFIG. 1. (While light can be directed onto the surface 11 from above, asshown in FIG. 1, it can also be incident on the photonic crystal frombelow and the same resonant effects are produced in the photoniccrystal).

The gain in sensitivity in the photonic crystal sensor 10 of FIG. 1obtained by (1) evanescent resonance (enhanced fluorescence due to oneresonance mode of the photonic crystal overlapping the excitation orabsorption spectrum of the fluorophore) and (2) enhanced extraction (dueto another resonance mode of the photonic crystal overlapping theemission spectrum of the fluorophore) are multiplied together to derivethe overall gain in sensitivity resulting from the combination of thesefeatures. For example, if the evanescent resonance provides for ahundred-fold increase in the amount of fluorescence emission, and theenhanced extraction provides the ability of the detector to collect afive times as many fluorescent photons, then the overall sensitivity ofan assay performed on a photonic crystal surface using the combinedtechniques will be five hundred (500) times greater than the same assayperformed on an ordinary surface (e.g., microscope slide, microplate ormicrofluidic flow channel) which does not possess the photonic crystalproperties of this invention.

Thus, one benefit of a photonic crystal sensor 10 with the doublyresonant properties as described above is that it provides a sensorplatform in which a very substantial increase in the sensitivity offluorescent assays occurs than would otherwise be possible. As will beappreciated from the following detailed description, photonic crystalsensors in accordance with the invention are useful in a number ofdifferent applications. These include:

1) Gene expression microarrays incorporating the photonic crystal sensor10. The genes may be detected at lower expression levels and or withsmaller sample volumes than previously known.

2) Protein detection assays, such as for example detection of proteinbiomarkers in bodily fluids for disease diagnostic tests, where proteinsare present in very low concentrations. Detection by the methods of thisdisclosure would be more sensitive than commonly use ELISA assays, butwith a simpler assay protocol.

3) Fluorescent imaging of cells, viruses, tissue samples, bacteria,proteins etc., using a microscope. The photonic crystal sensor 10 ofthis disclosure can be incorporated onto the surface of a microscopeslide. A specimen is stained with one or more fluorescent dyes orquantum dot fluorophores (which may be conjugated to an antibody orother probe of interest) and placed on the slide such that the specimenand fluorophore(s) are in contact with the surface of the photoniccrystal sensor 10. The improved sensitivity can be used to observe dyemolecules at lower concentrations and/or to use lower cost imagingcameras with an improved signal to nose ratio.

4) The photonic crystal sensor 10 can be produced uniformly over largesurface areas using a nano-replica molding process which is suitable formass production at low cost. The photonic crystal structure thusproduced can be incorporated into the surface of various assay ortesting devices of conventional formats, such as, for example, (1)incorporation onto the surface of a microscope slide, (2) incorporationwithin a standard format microplate, e.g., at the bottom thereof, or (3)incorporation into any other fluorescent assay format either now knownor later developed.

Thus, in one embodiment of the invention, a sensor 10 is descried hereinwhich is adapted to test a sample having a fluorophore present in thesample which is deposited on the sensor surface. The fluorophore (e.g.quantum dot, fluorescent dye such as Cy5) has an excitation spectrum andan emission spectrum. The sample may be placed on the sensor 10 in a dryor an aqueous environment. The sensor includes a photonic crystal havinga periodic grating structure (12). The photonic crystal exhibits aplurality of resonant modes when illuminated with light at an incidentangle θ. The resonant modes include an excitation mode having a firstresonant spectrum and an extraction mode having a second resonantspectrum. The periodic grating structure is constructed and arrangedsuch that the first resonant spectrum of the photonic crystal in theexcitation mode at least partially overlaps the excitation spectrum ofthe fluorophore and the second resonant spectrum of the photonic crystalin the extraction mode at least partially overlaps the emission spectrumof the fluorophore.

In one embodiment, the extraction resonant mode of the photonic crystalat the incident angle exhibits a relatively low Q factor, i.e., one inwhich the Q factor is less than 100. The Q factor of the extraction modewill determine the rate at which coupled radiation will be scatteredinto free space, such as in the case where the fluorophore exhibits abroad emission spectrum. In other embodiments, the extraction resonantmode has a Q factor is relatively high, i.e., between 100 and 1000. Witha relatively high Q factor, a detector can obtain enhanced extractionfrom a narrow band of wavelengths, but with amplified extractionefficiency. A sensor constructed to produce the optimum Q factor at theextraction resonant mode for a given application will depend on severalfactors, such as

1) whether the sensor is designed to be used in a detection instrumentwhich performs single-point detection (with a photomultiplier tube, asan example) where one can gather a broad range of wavelengths with abroad range of exit angles, situations where a low Q factor for theextraction resonant mode might be best, and

2) whether the sensor is designed to be used in a detection instrumentwhich performs imaging detection (e.g., with a CCD camera), in whichcase a relative high Q factor would provide detection of a narrow rangeof wavelengths and a narrow range of exit angles for that wavelength.The Q factor for the extraction resonance mode can be changed bychanging the parameters of the surface grating structure and simulatedin the design phase as will be described below.

The sensor as described herein can be incorporated into testingplatforms suited to a variety of specific applications. In one example,the sensor is incorporated into a gene expression microarray device. Inanother example, the sensor is incorporated into a protein detectionassay device and the sample is in the form of a protein. In still otherexamples, the sensor is incorporated into a testing device, such as amicroscope slide, which is used to perform fluorescent image analysis ofcells, viruses, bacteria, spores, and tissue samples. In still anotherexample the sensor is incorporated into a microwell plate having aplurality of individual sample wells. Each of the sample wells includesa photonic crystal as described herein.

In one particular embodiment, the periodic surface grating structure ofthe photonic crystal is constructed as a grating layer arranged as atwo-dimensional array of holes each having a depth D, and a relativelyhigh index of refraction material of thickness t deposited on thegrating layer. Suitable high index of refraction materials includetitanium oxide (TiO₂), silicon nitride, hafnium oxide, zinc sulfide,tantalum oxide and zinc selenide. In preferred embodiments the array ofholes has an axis of symmetry which is either perpendicular or parallelto the polarization state of the incident light. In FIG. 2 a, the linesconnecting the features Γ, X and M are axis of high symmetry in thephotonic crystal surface. The grating layer is positioned above asubstrate layer. The substrate layer may consist of a layer of glass,quartz, polymers, plastic, polyethylene terepthalate (PET) andcombinations thereof.

The depth D of the holes can be selected such that the photonic crystalexhibits the excitation and extraction resonance modes having spectrawhich are spectrally separated from each other and which substantiallyoverlap the excitation and emission spectra, respectively, of apredetermined fluorophore, such as a particular quantum dot or group ofquantum dots or other fluorophores having similar excitation andemission spectra.

In another aspect of this invention, a sample testing system isdescribed for testing a sample having a fluorophore present in thesample which is deposited on the photonic crystal sensor. Thefluorophore has an excitation spectrum and emits fluorescence in anemission spectrum. The sample testing system includes a detectioninstrument comprising a light source and a detector, and a photoniccrystal sensor comprising a periodic grating structure. The sampleincluding the fluorophore are placed on the periodic grating structure.

The particular construction of the detection system is not particularlyimportant and can vary widely, depending on the particular application.Examples of suitable detection systems include those systems describedin U.S. Pat. Nos. 7,118,710, 7,094,595, and 6,990,259 and U.S. Publishedapplications 2007/0009968; 2002/0127565; 2003/0059855; 2007/0009380; and2003/0027327.

The light source of the detection system is oriented relative to thephotonic crystal sensor such that the light source illuminates thephotonic crystal sensor at a incident angle θ in which the photoniccrystal simultaneously exhibits a plurality of resonant modes, theresonant modes including an excitation mode having a first resonantspectrum and an extraction mode having a second resonant spectrum.

The periodic grating structure is constructed and arranged such that thefirst resonant spectrum of the photonic crystal in the excitation modeat least partially overlaps the excitation spectrum of the fluorophoreand wherein the second resonant spectrum of the photonic crystal in theextraction mode at least partially overlaps the emission spectrum of thefluorophore.

In one embodiment, the detector may take the form of an imagingdetector, e.g., charge coupled device (CCD) camera. Other types ofdetectors are also possible, including photomultipliers. In preferredembodiments the light source may be a laser or a broad spectrum source.The light from the source may be polarized. In one embodiment, thegrating structure of the photonic crystal has an axis of symmetry whichis substantially parallel or perpendicular to the polarization state ofthe incident light.

The sensor can be incorporated into a variety of different testingdevice formats, as explained herein, such as a gene expressionmicroarray device, a protein detection assay device, a microscope slide,and a microwell plate or dish having a plurality of individual samplewells, in which each of the sample wells includes a photonic crystaldescribed herein.

In a further aspect, a method is disclosed of testing a sample having afluorophore bound to the sample, the fluorophore having an excitationspectrum and an emission spectrum, comprising the steps of: (a) placingthe sample onto the surface of a photonic crystal sensor, (b)illuminating the photonic crystal biosensor with light at an angle ofincidence θ, the biosensor responsively and simultaneously exhibiting(1) an excitation resonance mode having a spectrum which at leastpartially overlaps the excitation spectrum of the fluorophore; and (2)an extraction resonance mode having a spectrum which at least partiallyoverlaps the emission wavelength distribution of the fluorophore, theillumination and the resulting excitation and extraction resonance modescausing the fluorophore to emit light, and (c) collecting the emittedlight from the fluorophore and directing the emitted light onto adetector.

Example

FIG. 2 a is an illustration of a photonic crystal sensor 10 inaccordance with one embodiment of the invention. The sensor 10 includesa glass substrate layer 22, a grating layer 24 providing a periodicgrating structure (in this case a two dimensional array of holes 12) anda high index of refraction layer 26 of TiO₂ which is deposited on thegrating layer 24.

In order to design a PC that can support multiple guided-moderesonances, a two-dimensional structure with a sufficiently largeeffective index and the features arranged as a square lattice of holeswas chosen, as shown in FIG. 2 a. The period (Λ) of the structure waschosen such that it supports a relatively high Q-factor resonant modesat a wavelength where the fluorophores (in this instance, quantum dots)are excited (λ=488 nm, excitation mode) and low Q-factor modesoverlapping the quantum dot fluorescence emission spectrum (centered atλ=616 nm, extraction mode). In FIG. 2 a, the features Γ, X and M arepoints of high symmetry in the photonic crystal surface. Λ=300 nm is theperiod of the surface grating structure (with a square unit cell in Xand Y directions) and t=125 nm, the thickness of a high index ofrefraction material layer 26 which is deposited onto the grating layer24. Θ is the angle the incident beam of light 13 makes with the vertical(direction normal to the surface 11).

The logic governing the choice of low Q-factor extraction modes for someembodiments will become clear shortly, and although a relatively lowQ-factor resonance mode at the emission spectrum is shown (Q<100) inother applications a relatively high Q-factor (100≦Q<1000) may bedesired. The depth “D” of the holes 12 was chosen to provide therequired spectral separation between the excitation and extractionmodes. The thickness ‘t’ of the TiO₂ high index layer 26 was chosen tofine tune the spectral location of the resonant modes. The photoniccrystal sensor of FIG. 1 a was cost-effectively fabricated by anano-replica molding approach in an asymmetric configuration, so as toprovide the required mechanical stability and maintain a simplisticfabrication procedure. A combination of high refractive index (RI)material (layer 26) and low refractive index material (layer 24,“Nanoglass”™, Honeywell) was important in order to provide sufficienteffective index and positioning of the modes, respectively. Using a lowRI material for layer 24 allows the modes to be positioned closer to thedevice's upper surface 11, whereas a high RI material for layer 24 whichwould draw the modes deeper into the device 10 and subsequently reducetheir interaction with the fluorophores. The top surface 11 and crosssection SEM images of a fabricated model device are shown in FIG. 2 b.The selection of parameters of the device (refractive index, thickness,periodicity, etc.) can be chosen to provide the desired location ofresonant modes, as further described herein.

While an array of holes is shown in the embodiment of FIG. 2 a, othertypes of periodic structures are possible. Such structures generallyhave a configuration of periodic high and low regions (referred toherein occasionally as “grooves”), which can take a variety of forms. Inone embodiment, the thickness of the high index layer 26 is in the range30 to 1000 nm, e.g. 50 to 300 nm, preferably 50-200 nm. The period ofthe corrugated structure may be in the range 200 to 1000 nm, e.g. 200 to500 nm, preferably 250-500 nm. The ratio of the groove depth to thethickness of the high index layer 26 lies in the range 0.02 to 1 e.g.0.25 to 1, preferably 0.3 to 0.7, and the ratio of the grooves width tothe period of the grooves (“duty-cycle”) lies in the range 0.2 to 0.8,e.g. 0.4 to 0.6. Increasing the thickness of the high refractive indexlayer 26, the refractive index of the layer 26, the refractive index ofthe low index periodic grating layer 24, or the period of the gratingwill tend to increase the wavelength of the resonant mode.

The grooves may be generally rectangular in cross-section.Alternatively, the grooves may be sinusoidal or of saw toothcross-section. The surface structure may be generally symmetrical.Preferred geometries include rectangular, sinusoidal and trapezoidalcross-sections. Alternatively, the grooves may be of saw toothcross-section (blazed grating) or of other asymmetrical geometry. Inanother aspect the groove depth may vary, e.g. in periodic modulations.

The support or platform may be square or rectangular and the grooves mayextend linearly along the platform so as to cover the surface.Alternatively the platform may be disc shaped and the grooves may becircular or linear.

The grating structure can take variety of one and two dimensional forms,including two-level, two dimensional gratings, as disclosed in publishedPCT application WO 2007/0179024, the contents of which are incorporatedby reference herein. These include square lattices and hexagonallattices that have symmetry in three directions along the planar surfaceof the structure.

The corrugated, periodic grating surface may be optimized for oneparticular excitation wavelength and for one particular type ofpolarization. By appropriate means, e.g. superposition of severalperiodic structures which are parallel or perpendicular one withanother, periodic surface relief can be obtained that are suitable formultiple wavelength use of the photonic crystal sensor (“multicolor”applications). Alternatively, individual sensing areas on one platformmay be optimized for different wavelengths and/or polarizationorientations.

In another embodiment, the photonic crystal is constructed so as toexhibit a first extraction resonance mode in a first spatial area of thephotonic crystal and a second extraction resonance mode in a secondspatial area of the photonic crystal distinct from the first spatialarea. In other words, the construction of the surface grating structurecan vary spatially (in X and Y directions) such that different areas ofthe photonic crystal exhibit different extraction resonant modes. Thisspatial pattern of different extraction modes can be repeating. Thefirst extraction resonance mode has a spectrum at least partiallyoverlapping the emission spectrum of a first predetermined fluorophore(e.g., a particular quantum dot) and wherein the second extractionresonance mode has a spectrum at least partially overlapping theemission spectrum of a second predetermined fluorophore different fromthe first predetermined fluorophore (e.g., a second quantum dot).

Additionally, the construction of the surface grating structure 12 canvary spatially (in X and Y directions) such that different areas of thephotonic crystal exhibit different extraction resonant modes. As anexample, the photonic crystal exhibits different excitation resonantmodes at different spatial regions on the surface so that thefluorescent dye Cy5 is excited in one location; and the fluorescent dyeCy3 is excited in another nearby location. One could alternate between agroup of fluorophores (red, green, blue) much like how a single pixel ofa video display is comprised of multiple color emitters arranged closeto each other.

In another possible embodiment the photonic crystal is constructed so asto exhibit a plurality of extraction resonance modes, each of theplurality of extraction resonance modes having a resonant spectrum atleast partially overlapping an emission spectrum of a differentpredetermined fluorophore.

Enhanced Excitation

When externally incident light 13 (FIG. 2 a) interacts with periodicallymodulated structures (holes 12) in the sub-wavelength regime for aphotonic crystal, only the 0^(th) order forward and backward diffractedwaves can propagate. The periodicity however, also allows forphase-matching of higher (evanescent) orders to localized leaky modessupported by the photonic crystal 10. Once excited, the leaky modes,defined by a complex propagation constant, possesses a finite lifetimeas they are leaked out both in the forward (transmitted) and backward(specular) directions. The backward reradiated waves are in phase andconstructively interfere with the 0^(th) backward diffracted order whilethe forward reradiated waves are out of phase with the 0^(t) ^(h)forward diffracted order by π radians, causing destructive interferenceand consequently resulting in zero transmission. Thus, the externalexcitation of the leaky modes by means of incident light 13 (FIG. 2 a)is associated with a 100% reflection phenomenon for the resonantwavelength, assuming a defect-free, lossless system. Since the excitedleaky modes are radiative but localized in space during their finitelifetimes, they can be engineered to have very high energy densitywithin regions of the photonic crystal at resonance. The magnitude ofthis energy density is directly related to the resonant mode lifetime orQ-factor of resonance, which in turn can be controlled by adjusting thedevice parameters (thickness, refractive index, depth of holes,periodicity, etc.). Therefore, the intensity of emission of afluorophore (14, FIG. 1) (which is absorptive at the resonantwavelengths) can be greatly enhanced by placing the fluorophores inproximity to regions where the resonant modes concentrate most of theirenergy. In the example of FIG. 2A, this region is the bottom of theholes, as shown in FIG. 4 a, as will be explained below.

Enhanced Extraction

Concurrently with the enhanced excitation as just described, theexistence of leaky modes in the photonic crystal 10 that overlap thefluorescence emission spectrum opens up additional pathways for theemitted light to escape into free-space. Besides direct emission, thefluorescence can couple to the overlapping leaky modes and Bragg scatterout of the photonic crystal sensor, thereby greatly reducing the amountof light trapped as guided-modes, in comparison to an un-patternedsubstrate (as explained above in FIG. 1). If the dispersion of theseoverlapping emission leaky modes is close to the Γ-point band-edge, i.e.K₁₁ (magnitude of in-plane wave vector)˜0, a significant amount of theemitted light will be extracted within small angles with the vertical.It can thus be appreciated that enhancement of fluorescence can beachieved by enhanced excitation and enhanced extraction acting inconcert together at the same time.

Results for Example 1

Studying the reflection/transmission properties of a photonic crystal isa convenient technique to map out the dispersions of the leaky modes.Rigorous Coupled-Wave Analysis (RCWA) techniques were used to simulatehow the device of FIG. 2 a would respond in transmission to externallyincident radiation.

FIG. 3 a shows the computed leaky mode band structure, i.e. spectrallocation and transmission efficiency of the resonances as a function ofthe angle of incidence (θ) of light 13 (FIG. 2 a), along the Γ-Mdirection. As θ is increased from 0°, K_(∥) begins to increase andresults in degenerate resonances to separate into their respectiveconstituent orders, as indicated at 40 in FIG. 3 a. Experimentalverification of the band structure of the fabricated device was carriedout by mounting the device in a linear transmission setup, illuminatingit with collimated white light and plotting the resulting transmittedspectrum as a function of θ, results of which are shown in FIG. 3( b).Excellent qualitative agreement between simulation and experiment in the460 nm 500 nm range was seen, where the RIs of the materials varyslowly. Theoretically, it was predicted that the resonance at λ=488 nmshould occur at θ=11.2° (the resonance indicated the region 42 in FIG. 3a), and this is accurately observed in experiment. At shorterwavelengths, the RI of TiO₂ begins to increase divergently (n_(TiO2)=2.7at λ=400 nm) and is considerably less at longer wavelengths(n_(TiO2)=2.36 at λ=600 nm), leading to deviations from simulationsassuming a constant RI (n_(TiO2)=2.46 at λ=488 nm). This is clearly seenin the theoretically predicted higher order bands originating fromshorter wavelength resonances and red-shifted longer wavelength bands,which experimental results do not agree with. For the excitation modeshown in FIG. 3A, the Q-factor of resonance was found to be ˜155. Therelatively high Q-factor is indicated in FIG. 3 a by the thin, highlydefined line 41 of zero transmission efficiency in the region 42.

FIGS. 4 a and 4 b show the simulated electric near-field intensity (E²)(normalized to the unit amplitude incident field) at the two availablesurfaces of the device, for the excitation of the resonant mode at λ=488nm. FIG. 4 a shows the intensity at the bottom of the holes 12 of FIG. 2a. FIG. 4 b shows the intensity at the upper surface 11 of the sensor10. The influence of the resonance phenomenon on the resultingnear-fields is clearly seen as manifested in the enhanced electric fieldintensity. Similar enhancement can also be seen for the magneticnear-fields. It is apparent that for the lower available surface (FIG. 4a) (bottom of the hole) the excitation mode concentrates its energywithin the cavity region (the term cavity is used here strictly inrelation to the shape of the cross-section). At the upper surface (FIG.4 b), the energy is concentrated at the cavity periphery and beyond.Both the bottom of the well and the areas on the surface 11 adjacent tothe holes are where the fluorophore will be present during use, hencethe device exhibits strong intensities in the excitation resonance modein the areas of interest. Above the surfaces shown in FIGS. 4 a and 4 b,the field intensity decays exponentially (as previously shown). Inpractice, with a finite fluorophore which is not at the surface andunavoidable losses, the exact near-field intensity available to thefluorophore will always be lower than shown in FIGS. 4 a and 4 b.

The amount of amplification for enhanced excitation detection is relatedto the power transferred from the device structure to a distribution offluorophores on the sensor surface at the excitation wavelength of thefluorophore. The power density distribution of the sensor surface at theresonant wavelength, provided that the resonant wavelength is matched tothe excitation wavelength, therefore provides a means for comparing thesensitivity of different device designs. One can define the crossproduct E (max)×H (max) as a field power or “magnification factor”.While a more thorough analysis of the intensity distribution of theevanescent field from the tops, bottoms, and sides of the structure, anda detailed integration of power density to account for differencesbetween higher and lower power regions would provide a more exactprediction of whether one device will function more effectively thananother, the product of the maximum magnitude of an E component with anorthogonal H component provides a very simple, rough way of comparingdesigns. Nevertheless, studying the near-field intensity at theavailable surfaces gives a convenient metric to optimize the photoniccrystal design. In this case, it is also important to mention that dueto the inherent asymmetry of the photonic crystal, the mode concentratesits energy in the high index layer (26) and is biased more towards thegrating layer below it. By reversing the asymmetry (that is, by floodingthe device surface with higher index material, such as water) we canreverse this biasing and to an extent, ‘draw’ the resonance mode closerto the device surface, therefore further increasing the mode interactionwith the fluorophore. Such a modification will be easily adaptable forenhanced fluorescence biosensors, for example, where the analytes boundto the fluorophores are typically in an aqueous buffer solution.Near-field scanning optical microscopy (NSOM) images of the fabricateddevices excited close to the resonant wavelength have confirmedenhancement and spatial localization of the electric field intensity.

The effect of the leaky modes that overlap the emission spectrum of thefluorophores, which as per design, provide maximum overlap close to theΓ-point band-edge, will now be discussed. Such a choice is easilyjustified for maximal near-vertical extraction. In this enhancedextraction phenomenon, the Q-factor of the extraction modes willdetermine the rate at which coupled radiation will be scattered intofree-space. A low Q-facfor (implying a short mode lifetime) would bebeneficial in some applications, as the coupled radiation can bescattered faster and thus the interaction of the radiation with lossesin the system can be limited. A low Q-factor is also desirable from thestandpoint that the radiation emitted from the fluorophores has a finitebandwidth, and a broad leaky resonance can scatter more of the emittedwavelengths in a given direction. Since the polarization and directionsof the emitted fluorescence for a fluorophore (e.g., quantum dot) infree-space can be assumed to be arbitrary, the various available leakymodes that can interact with the emitted light are considered. Theexperimentally determined dispersion of the leaky modes supported by thephotonic crystal in the Γ-X and Γ-M directions and for orthogonalpolarizations (S and P) is shown in FIGS. 5 a, 5 b and 5 c. (If anelectromagnetic wave is propagating toward a sensor surface at an angle,it will have two orthogonal components of electric field. The “S”component is the one with the electric field vector oriented parallel tothe sensor surface (so one can think of “S” standing for “skim”, sincethe electric field vector skims the surface), whereas the “P” componentis the electric field component with the vector oriented directly intothe sensor surface, and one can think of P as standing for “Plunge” asthe electric field vector plunges into the sensor surface. Incidentlight can have both components at the same time.)

The case involving Γ-M and S polarization is already shown in FIG. 3(a). It is clearly seen that the QD fluorophore whose emission spectrumis (centered at λ=616 nm) can couple to leaky modes supported by thephotonic crystal and be extracted out of the device, because thephotonic crystal has a resonance mode indicated by the line 45 in FIGS.5 a and 5 c which includes a region of resonance indicated at 47 whichincludes λ=616 nm at the angle of incidence 11.5 degree. Fromexperiment, the Q-factor for the extraction modes was ˜92.

To quantify the effects of the two fluorescence enhancement schemes, thefabricated devices were cleaned using de-ionized water/isopropylalcohol, and the QDs (CdSe/ZnS core-shell type, Evident Technologies,peak emission at λ=616 nm) were diluted in toluene and made up to aconcentration of 1.235 nM. The dilute solution of QDs was dropcast onand off (to serve as a reference) the photonic crystal surface. Afterthe drying of the spots, the devices were scanned on a commerciallyavailable laser scanner (LS 2000, Tecan), equipped with a 25 mW, 488 nmsolid-state laser and a photo multiplier tube (PMT) to record thefluorescence signals. The scanner provides the ability to launch theincident illumination at angles tunable from 0° to 25° in steps of 0.1°,along a single vertical axis and single polarization. In order, toquantify the extraction enhancement and the excitation enhancementeffects, the devices were scanned at the resonant angle (θ=11.2°) and anon-resonant angle (θ=0°). For the sake of clarity, the resonant angleis defined as the launch angle (θ) for which the leaky mode at λ=488 nmis excited. FIGS. 6 a and 6 b show the scanned images taken at θ=11.2°and θ=0° respectively. The circular regions 50 on the images show theareas over which intensity information was averaged. Table 1 shows theraw data measured in PMT counts for the two cases over multiplemeasurements. “PC” in Table 1 indicates “photonic crystal.”

TABLE 1 Signal Background On PC (S1) off PC (S2) on PC (B1) off PC (B2)θ = 11.2° 10,160.93 ± 362.92 131.78 ± 2.91 336.03 ± 9.14 41.52 ± 1.13 θ= 0°  705.31 ± 8.80  72.53 ± 0.93  25.33 ± 0.89 21.04 ± 0.48

The enhancement (calculated by (S1−B1)/(S2−B2)) for θ=11.2° was108.89±4.09, and for θ=0° was 13.21±0.26. The observed enhancement atθ=0° is attributed mainly to the enhanced extraction provided by thephotonic crystal. Since the extraction effect is only related to thedispersive properties of the photonic crystal, it should not be affectedby a change in the launch angle of the incident light. Using thisassumption, the total enhancement obtained for the resonant angle isdivided by the enhancement obtained for the non-resonant angle and avalue of 8.24±0.36 times for fluorescence enhancement by the near-fieldswas obtained.

DISCUSSION

The result for fluorescence enhancement due to the enhanced near-fields,at first glance, is much lesser than the peak intensity of thenear-fields shown in FIGS. 4 a and 4 b. However, one could expect thatthe spatially-averaged near-field enhancement would be much lower due tothe specific pattern of the field distributions, and more important indeciding the resulting enhancement due to the nonspecific positioning ofthe QDs. Furthermore, it was found that due to the inherent absorptionand fluorescence at visible wavelengths of most materials used in suchfabrication processes results in additional loss to the resonance. Inthe fabricated device, the combination of the spin-on glass material(Nanoglass™, Honeywell) for the grating layer and TiO₂ for the highindex layer was strongly absorbing and fluorescent at the excitationwavelength. This can be seen by the enhancement of the background signalfrom regions on the photonic crystal where no quantum dots are present(FIG. 6( b)). Indeed, the enhanced fields produced due to the resonanceeffect serve also to boost the background signal over 13 times,presenting a strong, undesirable loss mechanism that reduces theexcitation intensity available to the QDs. Alternative fabricationmethods and material choices can be used to minimize such losses, asknown in the art.

For the enhanced extraction case, the fluorescence enhancement isbelieved to be mostly related to Bragg scattering. The structurefabricated in this example, due to its asymmetric nature, cannot possesa bandgap for either TE or TM-like modes and therefore, the effect ofinhibited spontaneous emission into undetectable waveguide modes isabsent. Time-resolved fluorescence measurements on the QDs both on andoff the photonic crystal surface helped verify the absence of cavityenhanced spontaneous emission via the Purcell effect (data not shown).Enhanced extraction was verified by angle-resolved fluorescencemeasurements. By illuminating the photonic crystal and measuring thefluorescence emitted by the QDs at different angles, the enhancedextraction phenomenon was verified and shows strong coupling between theextraction modes and the QDs. The extraction effects may be furtheroptimized by reducing the anisotropy of the in-plane wave vector, byemploying photonic lattices whose Brillouin zones are more circular,e.g. triangular or quasi-periodic lattices. The density of extractionmodes will also affect the extraction efficiency. A greater density ofmodes that overlap the emission spectrum of the QDs, would result instronger scattering and consequently extraction effects. Finally, byengineering the spectral overlap and dispersion properties of thevarious leaky modes supported by the photonic crystal one can extend theenhancement effect to a wide range of fluorescent species.

Such a fluorescence enhancement scheme can be invaluable to theapplication of fluorescent biosensing using QDs, for example. Given theexcellent applicability of QDs to serve as fluorescent probes, a highlysensitive fluorescence detection system is provided that will enableworking at very low/single molecule analyte concentrations. Such adetection scheme will inherently incorporate low backgroundfluorescence, as the QD tags close to the biosensor surface willexperience maximum fluorescence enhancement, similar to total internalreflection fluorescence (TIRF) microscopy.

Here, we have demonstrated resonant enhancement of over 108 times influorescence from QDs on the surface of a 2D photonic crystal. This hasbeen achieved by engineering the photonic crystal such that it possessesleaky eigenmodes (resonance mode) at the absorption and emissionwavelengths of the QDs. The results of this work can be adapted to awide variety of optical applications involving QDs, including highbrightness LEDs, optical switches and high sensitivity biosensors.

Fabrication Methods

The two-dimensional photonic crystals described herein can be fabricatedby a nano-replica molding process, described in the previously citedpatent literature. Briefly, electron beam lithography (JEOL JBX-6000FS)was used to define a two-dimensional ‘square lattice of holes’ surfacestructure of period Λ=300 nm and hole radius r=90 nm on a SiO₂/Sisubstrate with PMMA as the mask layer. The pattern was exposed to a sizeof 3×3 mm² followed by development and dry etching in a CHF3 reactiveion etching process. The resulting surface structure was subsequentlytransferred to a glass substrate (PET film or glass), coated with alow-index porous spin-on-glass (Nanoglass, Honeywell) using anintermediate polydimethylsiloxane (PDMS) stamp. A thin layer of highindex TiO₂ (t=125 nm) was then sputtered (AJA International Inc.) toform the final device. The refractive indices (RI) of the Nanoglass (ng)and TiO₂ materials as determined by spectroscopic ellipsometry (Woolam)were n_(ng)=1.17 and n_(TiO2)=2.46 respectively at λ=488 nm.

Simulation and Device Design

A commercial implementation of the RCWA code (GSolver) was employed forall the simulations. One period of the device was simulated, withperiodic boundary conditions applied to the x and y extents. Theincident radiation was set to be S-polarized plane waves incident fromabove the device and along the Γ-M direction (θ=45°, the choice of theselaunch parameters were essentially dictated by limitations of ourexperimental setup). To improve the calculation speed for the leaky modeband structure, the materials were assumed lossless and the RIdispersion was assumed to be flat about λ=488 nm. Near-fieldcalculations however, were performed including the complex component ofthe material refractive indices (k_(NG)=0 and k_(TiO2)=0.00036) andretaining 12 harmonics in both the x and y directions.

NSOM Measurements of Fabricated Devices and Near-Field Enhancement.

Photonic crystal (PC) slab devices fabricated by nano-replica moldingwere inspected using the Witec Alpha near-field scanning microscope(NSOM). The devices were excited with λ=488 nm excitation from anargon-ion laser and the near fields were probed close to resonance. Theresulting near-field intensity map is shown in FIG. 7 and shows adistribution qualitatively identical to the simulated near-field in FIG.4( b), within the limited lateral resolution of the NSOM.

Enhancement of the near-field is also evident in the NSOM image of FIG.7. Defects incorporated into the device during fabrication are clearlyvisible as regions where the nearfield distributions are distorted.

From FIGS. 4 a and 4 b one can predict the absolute maximum enhancementobtainable by spatially averaging the predicted near-field distributionsover the device surface. For the distributions relevant to the designeddevices, a maximum average intensity of ˜240 times is calculated at thedevice surface. However due to unavoidable resonator losses and finitesize of the quantum dots (˜5 nm), the practical enhancement in theexcitation intensity would be lower.

Experiments Verifying the Enhanced Extraction Provided by the PhotonicCrystal.

In order to verify the enhanced extraction effect of the photoniccrystal due to the overlap of its leaky eigenmodes with the fluorescencespectrum of the QDs, angle-resolved fluorescence measurements wereperformed. The QDs were used at a 100× higher concentration (123.5 nM)for this experiment to provide sufficient signal for detection. Using ahigher concentration resulted in the band structure of the leaky modesbeing slightly red-shifted, due to the increase in effective-index forthe resonances. Consequently, this results in slightly increased anglesfor extraction.

The enhanced extraction phenomenon is believed to occur when the leakymodes (termed extraction modes) of the photonic crystal overlap with theQD fluorescence emission spectra. This overlap creates ‘channels’ intowhich the QDs can couple their energy. Since the modes are by definitionleaky, this coupled emission from the QDs must also leak intofree-space. The direction (angles) of leakage will also follow thedispersion of the extraction modes. In order to extract all the emittedlight in a single direction, the bandwidth of the leaky mode to shouldbe equal to or larger than the fluorescence bandwidth. This has beenexplained as the rationale behind designing photonic crystals with arelatively low Q-factor extraction modes.

In experiment, the photonic crystal containing the QDs on its surfacewas mounted on a fixed stage. Incident light from a λ=488 nm, 10 mWargon-ion laser was normally incident on the device, and provided therequired excitation for the QDs. It must be noted that the incidentlight is not resonant with the photonic crystal at this angle, andtherefore the near-fields are not enhanced. The fluorescence wasdetected from the sample by a fiber probe set at a distance of L=10 cmfrom the device center. A band stop filter filtered out the laserexcitation so that only fluorescence from the QDs was observed. Theprobe was rotated about the device and the fluorescence spectrum wascollected. By rotating the sample orientation, the spectrum Was recordedfor both the Γ-M and Γ-X directions.

FIG. 8 shows the angle resolved fluorescence spectrum as measured fromthe PC when the measurement is taken along the Γ-M direction. Since apolarizer is not used to filter the emitted radiation, all polarizationsof emitted radiation are detected in the same measurement. For the caseof the Γ-M direction, a broad angle-independent fluorescence featurethat ranges from 600-650 nm is seen, indicated by the lighter region100. This is the fluorescence from the QDs. Superimposed upon thisfeature, strong features that match the extraction mode band structureexperimentally determined in FIGS. 2 & 4 are seen, indicated by thebright regions of highest detected intensity in FIG. 8, indicated at102. This is a clear representation of the strong coupling between theQD fluorescence and the leaky modes of the PC. As seen, the fluorescenceis also enhanced when the leaky modes overlap the fluorescence spectrum,the strongest enhancement being obtained when the peak of the leaky modeoverlaps the peak wavelength of the fluorescence emission spectrum.

A different way of looking at the two-dimensional experimental datashown in FIG. 8 is shown in FIG. 9, where slices of data from FIG. 8 arenormalized and superimposed upon each other. In FIG. 8, the curve 104represents the emission of the quantum dots when no photonic crystal ispresent. The curve 106 represents the condition when the leaky mode justbegins to overlap the fluorescence spectrum of the quantum dots (atnormal incidence, θ=0°). A clear modification in spectralcharacteristics is seen, as in the appearance of a lower wavelength peak108 resulting from enhancement of the QD sideband emission. When thepeak of the emission matches the peak of the quantum dot emission(A=10°, dark square curve), a dramatic change in spectralcharacteristics of the emitted radiation involving emission bandwidthreducing to roughly half its original value is evident, indicated bycurve 110. Thus, the presence of the photonic crystal results in strongspectral and spatial modification of the fluorescence emitted by theQDs.

FIG. 10 shows the measurement of angle-resolved fluorescence for the Γ-Xdirection. In this case, two extraction bands 120 and 122 overlappingthe fluorescence spectrum 100 from the QDs are seen in comparison to oneas shown in FIG. 8. This is due to the fact that in the Γ-X direction,the leaky band structure for the extraction modes is different fordifferent polarizations of coupled light. Each of the bands 120 and 122appearing in FIG. 10 corresponds to either S-polarized or P-polarizedlight being emitted by the QDs. In the Γ-M direction, the dispersion ofthe extraction leaky modes is independent of the polarization. This isalso seen clearly in FIGS. 3 & 5, where the dispersions of theextraction modes for the Γ-M direction are same for both polarizations(S & P) but different for different polarizations in the Γ-X direction.

FIG. 11 is a graph of the intensity of quantum dot emission as afunction of time, showing that quantum dots decay significantly fasterwhen placed on either a TiO2 substrate or a photonic crystal as comparedto a glass slide.

While the above example has used a quantum dot fluorophore, theinvention is applicable to detection of any fluorophore orfluorescently-labeled group. Using a non-QD fluorophore (such as anorganic fluorescent dye) with the inventive photonic crystal sensor willstill provide significant fluorescence sensitivity enhancement.

Extraction Mode Only Sensors

While the above example has demonstrated a photonic crystal sensor withresonance modes which overlap both the excitation and emission spectraof a fluorophore, in another embodiment the photonic crystal can bestructured and arranged such that the photonic crystal exhibits aresonant mode when illuminated with light at an incident angle θ whichat least partially overlaps the emission spectrum of the fluorophore,but the photonic crystal does not simultaneously have a resonance modewhich overlap the excitation spectrum of the fluorophore. Such a sensorwould exhibit the enhanced extraction effect but not the enhancedexcitation effect. The sensor would be useful of many applications, suchas those described previously.

A sample testing system for testing a sample is envisioned using asensor featuring just the enhanced extraction mode. The system wouldinclude a detection instrument comprising a light source and a detector,and a photonic crystal sensor comprising a periodic grating structure,with the sample including the fluorophore being placed on the periodicgrating structure. The light source of the detection system is orientedrelative to the photonic crystal sensor such that the light sourceilluminates the photonic crystal sensor at a incident angle θ in whichthe photonic crystal exhibits a resonant mode having a resonant spectrumwhich at least partially overlaps the emission spectrum of thefluorophore. The detector operates to detect radiation from thefluorophore in the emission spectrum.

Yet further, in this embodiment, a method is provided for testing asample having a fluorophore bound to the sample. The method includes thesteps of placing the sample onto the surface of a photonic crystalsensor; illuminating the photonic crystal biosensor with light at anangle of incidence θ, the biosensor responsively exhibiting anextraction resonance mode having a spectrum which at least partiallyoverlaps the emission spectrum of the fluorophore, the illumination andthe resulting extraction resonance mode causing the fluorophore to emitlight; and collecting the emitted light from the fluorophore anddirecting the emitted light onto a detector.

Other Examples

Photonic Crystal Constructions

The substrate layer and grating layers of the photonic crystal sensormay be formed from inorganic materials such as glass, SiO₂, quartz,silicon, and of different organic and inorganic components or layers ascomposite materials. Alternatively the layers can be formed from organicmaterials such as polymers preferably polycarbonate (PC), poly (methylmethacrylate) (PMMA), polyimide (PI), polystyrene (PS), polyethylene(PE), polyethylene terepthalate (PET) or polyurethane (PU). Substratematerials also include polycarbonate or cyclo-olefin polymers such asZeanor®.

The high index of refraction layer on the top of the substrate may beformed from inorganic materials. Examples include metal oxides such asTa₂O₅, TiO₂, Nb₂O₅, ZrO₂, ZnO or HfO₂.

The embodiment of a two-dimensional grating structure suitable forsimultaneous fluorescence enhancement by enhanced fluorescenceexcitation and enhanced extraction is disclosed and may be preferred insome implementations. A two-dimensional grating can look like a waffle(holes), a waffle iron (posts), or a chessboard configuration withalternating high and low regions in two dimensions, for example.Two-dimensional gratings can have different periods in the X and Ydirections. These features may have various profiles in the Z directionsuch as angled or curved sidewalls. Thus, in the case of the wafflepattern, the impressions or wells may have a rectangular rather than asquare shape. This added flexibility provided by two dimensionalgratings allows one to tune the resonance positions for enhancedexcitation and extraction detection to occur at different wavelengths.As an example, the X periodicity can provide a sharp resonance at ornear normal incidence with wavelength tuned to excite the fluorophorewhile the Y periodicity can yield a broad resonance that coincides withthe emission wavelength of the fluorophore. In one particular example,the X periodicity provides a resonance tuned to excite a Cy3 fluorophorewith green light, while the Y periodicity gives a broad resonance thatcoincides with the emission wavelength of Cy3.

A single photonic crystal surface may be used to support, in parallel, alarge number of fluorescence assays in the form of an array of probes orcapture molecules that are deposited upon different locations. Eachprobe/capture molecule (the terms probes and capture molecules are usedinterchangeably herein) may contain individual and/or mixtures ofcapture molecules which are capable of affinity reactions. The shape ofan individual capture molecule may be rectangular, circular,ellipsoidal, or any other shape. The area of an individual captureelement may be any suitable area, such as between 1 μm² and 10 mm²,between 20 μm² and 1 mm² and in one embodiment, between 100 μm² and 1mm². The capture molecules may be arranged in a regular two dimensionalarray. The center-to-center (ctc) distance of the capture elements maybe any suitable distance, such as between 1 μm and 1 mm, between 5 μm to1 mm, and between 10 μm to 1 mm.

The number of capture elements per sensing region is between 1 and1,000,000, preferably between 1 and 100,000. In another aspect, thenumber of capture elements to be immobilized on the platform may not belimited and may correspond to the number of desired features underinvestigation e.g. the number of genes, DNA sequences, DNA motifs, DNAmicro satellites, single nucleotide polymorphisms (SNPs), proteins orcell fragments constituting a genome of a species or organism ofinterest, or a selection or combination thereof. In a further aspect,the platform of this invention may contain the genomes of two or morespecies, e.g. mouse and rat, or human and mouse.

Sensor Platforms

The photonic crystal structures of this disclosure may be produceduniformly over large surface areas using a nanoreplica molding process.After manufacture, the structure may be incorporated onto the surface ofmicroscope slides, within standard format microplates, or any otherconvenient assay format, including microarray formats. A microarrayformat typically includes a large number (e.g., 10,000, or 100,000) ofdistinct locations. Such locations are typically laid out in a regulargrid pattern in x-y coordinates. However, a microarray can be laid outin any type of regular or irregular pattern. For example, distinctlocations can define a microarray of spots of one or more specificbinding substances. A microarray spot can be about 50 to about 500 μm indiameter or any other suitable diameter. A microarray on a support to beused in this invention can be used by placing microdroplets of a sampleincluding one or more specific binding substances and fluorophores onto,for example, an xy grid of locations on a two-dimensional grating orcover layer surface. When the biosensor is exposed to a test samplecomprising one or more binding partners, the binding partners will bepreferentially attracted to distinct locations on the microarray thatcomprise specific binding substances that have high affinity for thebinding partners. Some of the distinct locations will gather bindingpartners onto their surface, while other locations will not.

One example of a microarray to be used in a method according to thepresent invention is a nucleic acid microarray, in which each distinctlocation within the array contains a different nucleic acid molecule. Inthis embodiment, the spots within the nucleic acid microarray detectcomplementary chemical binding with an opposing strand of a nucleic acidin a test sample.

The sensors described here can be used to sensitively analyze a varietyof analytes. Some examples of analytes that can be detected using thesensors and methods herein include, but are not limited to, one or more:proteins, peptides, DNA molecules, RNA molecules, oligonucleotides,lipids, carbohydrates, polysaccharides; glycoproteins, lipoproteins,sugars, cells, bacteria, viruses, candidate molecules and allderivatives, variants and complexes of these, which have a fluorescentlabel. Other fluorescent substances can be detected, as known in theart. Nanomaterials such as quantum dots or functionalized quantum dotsmay be used. Applications include gene expression microassays wheregenes may be detected at lower expression levels and/or with Y smallersample volumes. Other applications include protein detection assays,such as detection of protein biomarkers in bodily fluids for diseasediagnostic tests, where proteins are present at very low concentration.Detection by the method described in this invention would be moresensitive than commonly used ELISA assays, but with a simpler assayprotocol. In addition, fluorescent imaging of cells and proteins usingfluorescent microscopes can utilize the techniques presented here, wherethe improved sensitivity can be used to observe dye molecules at lowerconcentrations and/or to use lower-cost imaging came due to improvedsignal-to-noise ratio

Alternative Grating Structures

In one embodiment, a support to be used in a method of the inventionwill be illuminated with white light that will contain light of everypolarization angle. The orientation of the polarization angle withrespect to repeating features in a biosensor grating will determine theresonance wavelength. For example, a “linear grating” biosensorstructure consisting of a set of repeating lines and spaces will havetwo optical polarizations that can generate separate resonantreflections. Light that is polarized perpendicularly to the lines iscalled “s-polarized,” while light that is polarized parallel to thelines is called “p-polarized.” Both the s and p components of incidentlight exist simultaneously in an unfiltered illumination beam, and eachgenerates a separate resonant signal. A support structure can generallybe designed to optimize the properties of only one polarization (thes-polarization), and the non-optimized polarization is easily removed bya polarizing filter.

In order to remove the polarization dependence, so that everypolarization angle generates the same resonant reflection spectra, analternate structure can be used that consists of a set of concentricrings. In this structure, the difference between the inside diameter andthe outside diameter of each concentric ring is equal to about one-halfof a grating period. Each successive ring has an inside diameter that isabout one grating period greater than the inside diameter of theprevious ring. The concentric ring pattern extends to cover a singlesensor location—such as a microarray spot or a microtitre plate well.Each separate microarray spot or microtitre plate well has a separateconcentric ring pattern centered within it. All polarization directionsof such a structure have the same cross-sectional profile. Theconcentric ring structure must be illuminated precisely on-center topreserve polarization independence. The grating period of a concentricring structure is less than the wavelength of the resonantly reflectedlight. The grating period is about 0.01 micron to about 1 micron, in oneembodiment. The grating depth is about 0.01 to about 1 micron, in oneembodiment.

In another embodiment, an array of holes or posts are arranged toclosely approximate the concentric circle structure described abovewithout requiring the illumination beam to be centered upon anyparticular location of the grid. Such an array pattern is automaticallygenerated by the optical interference of three laser beams incident on asurface from three directions at equal angles. In this pattern, theholes (or posts) are centered upon the corners of an array of closelypacked hexagons. The holes or posts also occur in the centre of eachhexagon. Such a hexagonal grid of holes or posts has three polarizationdirections that “see” the same cross-sectional profile. The hexagonalgrid structure, therefore, provides equivalent resonant reflectionspectra using light of any polarization angle. Thus, no polarizingfilter is required to remove unwanted reflected signal components. Theperiod of the holes or posts can be about 0.01 μm to about 1 μm and thedepth or height can be about 0.01 μm to about 1 μm.

The detecting system may be arranged to detect luminescence such asfluorescence. Affinity partners can be labeled in such a way thatFörster fluorescence energy transfer (FRET) can occur upon binding ofanalyte molecules to capture molecules. The maximum of the luminescelabels can be used to modify capture elements, assayed molecules in theanalyte, or any other species, e.g. endogeneous/exogeneous controls,spacer molecules, primers, bio/materials, that interact with the sensorsurface.

The luminescence dyes used as markers may be chemically or physically,for instance electrostatically, bonded to one or multiple affinitybinding partners (or derivatives thereof) present in the analytesolution and/or attached to the platform. In case of naturally occurringoligomers or polymers such as DNA, RNA, saccharides, proteins, orpeptides, as well as synthetic oligomers or polymers, involved in theaffinity reaction, intercalating dyes are also suitable. Luminophoresmay be attached to affinity partners present in the analyte solution viabiological interaction such as biotin/avidin binding or metal complexformation such as HIStag coupling.

One or multiple luminescence markers may be attached to affinitypartners present in the analyte solution, to capture elementsimmobilized on the platform, or both to affinity partners present inanalyte solution and capture elements immobilized at the platform, inorder to quantitatively determine the presence of one or multipleaffinity binding partners.

The samples may be used either undiluted or with added solvents.Suitable solvents include water, aqueous buffer solutions, proteinsolutions, natural or artificial oligomer or polymer solutions, andorganic solvents. Suitable organic solvents include alcohols, ketones,esters, aliphatic hydrocarbons, aldehydes, acetonitrile or nitriles.

Solubilisers or additives may be included, and may be organic orinorganic compounds or biochemical reagents such asdiethylpyrocarbonate, phenol, formamide, SSC (sodium citrate/sodiumchloride), SDS (Sodiumdodecylsulfate), buffer reagents, enzymes, reversetranscriptase, RNAase, organic or inorganic polymers.

Fluorophores and Fluorescent Labels

While the above examples have used quantum dots as the fluorescentmolecule which is excited by incident radiation, other fluorophores canbe used in accordance with the inventive biosensor.

Transfluorospheres or derivatives thereof may be used for fluorescencelabeling, and chemiluminescent or electroluminescent molecules may beused as markers.

Luminescent compounds having luminescence in the range of from 400 nm to1200 nm which are functionalised or modified in order to be attached toone or more of the affinity partners may be used, including derivativesof: polyphenyl and heteroaromatic compounds, stilbenes, coumarines,xanthene dyes, methine dyes, oxazine dyes, rhodamines, fluoresceins,coumarines, stilbenes, pyrenes, perylenes, cyanines, oxacyanines,phthalocyanines, porphyrines, naphthalopcyanines, azobenzenederivatives, distyryl biphenyls, transition metal complexes e.g.polypyridyl/ruthenium complexes, tris (2,2′ bipyridyl) rutheniumchloride, tris(1,10-phenanthroline) ruthenium chloride, tris (4,7diphenyl-1,10-phenanthroline) ruthenium chloride andpolypyridyl/phenazine/ruthenium complexes, such asoctaethyl-platinum-porphyrin, Europium and Terbium complexes may be usedas luminescence markers, nanoparticles, microparticles, or any otherlight emitting species that can be excited by evanescent fields.

Suitable for analysis of blood or serum are dyes having absorption andemission wavelength in the range from 400 nm to 1000 nm. Furthermoreluminophores suitable for two and three photon excitation can be used.

Dyes which are suitable in this invention may contain functional groupsfor covalent bonding, e.g. fluorescein derivatives such as fluoresceinisothiocyanate. Also suitable are the functional fluorescent dyescommercially available from Amersham Life Science, Inc., Texas, andMolecular Probes Inc. Other suitable dyes include dyes modified withdeoxynucleotide triphosphate (dNTP) which can be enzymaticallyincorporated into RNA or DNA strands. Further suitable dyes includeQuantum Dot Particles or Beads (Quantum Dot Cooperation, Palo Alto,Calif.) or derivatives thereof or derivatives of transition metalcomplexes which may be excited at one and the same defined wavelength,and derivatives show luminescence emission at distinguishablewavelengths.

Analytes may be detected either via directly bonded luminescencemarkers, or indirectly by competition with added luminescence markedspecies, or by concentration, distance, pH, potential- or redoxpotential-dependent interaction of luminescence donors andluminescence/electron acceptors used as markers bonded to one and/ormultiple analyte species and/or capture elements. The luminescence ofthe donor and/or the luminescence of the quencher can be measured forthe quantification of the analytes.

In the same manner affinity partners can be labeled in such a way thatelectron transfer or photoinduced electron transfer leads to quenchingof fluorescence upon binding of analyte molecules to capture molecules.

Luminescent labels can be used to modify capture elements, assayedmolecules in the analyte, or any other species, e.g.endogeneous/exogeneous controls, spacer molecules, primers,bio/materials, that interact with the sensor surface.

The luminescence dyes used as markers may be chemically or physically,for instance electrostatically, bonded to one or multiple affinitybinding partners (or derivatives thereof) present in the analytesolution and/or attached to the platform. In case of naturally occurringoligomers or polymers such as DNA, RNA, saccharides, proteins, orpeptides, as well as synthetic oligomers or polymers, involved in theaffinity reaction, intercalating dyes are also suitable. Luminophoresmay be attached to affinity partners present in the analyte solution viabiological interaction such as biotin/avidin binding or metal complexformation such as HIStag coupling.

One or multiple luminescence markers may be attached to affinitypartners present in the analyte solution, to capture elementsimmobilized on the platform, or both to affinity partners present inanalyte solution and capture elements immobilized at the platform, inorder to quantitatively determine the presence of one or multipleaffinity binding partners.

Capture Molecules Bound to Fluorophores

The fluorophores described herein (e.g., quantum dots) arefunctionalized by being bound to capture molecules which are in turndeposited onto the surface of the photonic crystal biosensor. The natureof the capture molecules are many and varied. Generally speaking thecapture molecules used should be capable of affinity reactions. Examplesof capture molecules which can be used in the context of this inventioninclude: nucleotides, oligonucleotides (and chemical derivativesthereof) DNA (double strand or single strand) a) linear (and chemicalderivatives thereof) b) circular (e.g. plasmids, cosmids, BACs, ACs),total RNA, messenger RNA, cRNA, mitochondrial RNA, artificial RNA,aptamers, PNA (peptidenucleic acids) Polyclonal, Monoclonal,recombinant, engineered antibodies, antigenes, haptens, antibody FABsubunits (modified if necessary), proteins, modified proteins, enzymes,enzyme cofactors or inhibitors, protein complexes, lectines, Histidinelabeled proteins, chelators for Histidinetag components (HIStag), taggedproteins, artificial antibodies, molecular imprints, plastibodies,membrane receptors, whole cells, cell fragments and cellularsubstructures, synapses, agonists/antagonists, cells, cell organelles,e.g. microsomes, small molecules such as benzodiazapines,prostaglandins, antibiotics, drugs, metabolites, drug metabolites,natural products, carbohydrates and derivatives, natural and artificialligands, steroids, hormones, peptides, native or artificial polymers,molecular probes, natural and artificial receptors and chemicalderivatives thereof, chelating reagents, crown ether, ligands,supramolecular assemblies, indicators (pH, potential, membranepotential, redox potential), viruses, bacteria and a tissue sample froman animal or plant subject.

In biological applications, the sample can be for example, blood,plasma, serum, gastrointestinal secretions, homogenates of tissues ortumors, synovial fluid, feces, saliva, sputum, cyst fluid, amnioticfluid, cerebrospinal fluid, peritoneal fluid, lung lavage fluid, semen,lymphatic fluid, tears, or prostatic fluid.

The biosensor surface may include an adhesion promoting layer disposedat the surface of the optically transparent layer (high index ofrefraction layer) in order to enable immobilization of capturemolecules. The adhesion promoting layer may also comprise a microporouslayer (for example, ceramics, glass, Si) for further increasing assayand detection efficacy or of gel layers which either can be used asmedium for carrying out the capture element immobilization and sampleanalysis, thereby further increasing the assay and detection efficacy,or which allow separation of analyte mixtures in the sense of gelelectrophoresis. The platform may be formed with a plurality of sensingareas or regions, each having its own diffractive grooves.

In other words, immobilization of one or more probes/capture moleculesonto a biosensor surface can be performed so that a specific bindingsubstance will not be washed away by rinsing procedures, and so that itsbinding to binding partners in a test sample is unimpeded by thebiosensor surface. Several different types of surface chemistrystrategies have been implemented for covalent attachment of specificbinding substances to, for example, glass for use in various types ofmicroarrays and biosensors. These same methods can be readily adapted toa biosensor of the invention. Surface preparation of a biosensor so thatit contains the correct functional groups for binding one or morespecific binding substances is an integral part of the biosensormanufacturing process.

One or more specific binding substances can hence be attached to abiosensor surface by physical adsorption (i.e., without the use ofchemical linkers) or by chemical binding (i.e., with the use of chemicallinkers). Chemical binding can generate stronger attachment of specificbinding substances on biosensor surface and provide defined orientationand conformation of the surface-bound molecules. For instance, sometypes of chemical binding include, for example, amine activation,aldehyde activation, and nickel activation. These surfaces can be usedto attach several different types of chemical linkers to a biosensorsurface. While an amine surface can be used to attach several types oflinker molecules, an aldehyde surface can be used to bind proteinsdirectly, without an additional linker. A nickel surface can be used tobind molecules that have an incorporated histidine (“his”) tag.Detection of “his-tagged” molecules with a nickel-activated surface iswell known in the art (Whitesides, Anal. Chem. 68, 490 (1996)).

Immobilization of specific binding substances to plastic, epoxy, or highrefractive index material can be performed similarly to that describedfor immobilization to glass. However, the acid wash step can beeliminated where such a treatment would damage the material to which thespecific binding substances are immobilized. This is well known in theart.

For the detection of binding partners at concentrations less than about0.1 ng/ml, it is possible to amplify and transduce binding partnersbound to a biosensor into an additional layer on the biosensor surface.The increased mass deposited on the biosensor can be easily detected asa consequence of increased optical path length. By incorporating greatermass onto a biosensor surface, the optical density of binding partnerson the surface is also increased, thus rendering a greater resonantwavelength shift than would occur without the added mass. The additionof mass can be accomplished, for example, enzymatically, through a“sandwich” assay, or by direct application of mass to the biosensorsurface in the form of appropriately conjugated beads or polymers ofvarious size and composition. This principle has been exploited forother types of optical biosensors to demonstrate sensitivity increasesover 1500 beyond sensitivity limits achieved without mass amplification.See, e.g., Jenison et al., “Interference-based detection of nucleic acidtargets on optically coated silicon,” Nature Biotechnology 19: 6265(2001).

As an example, a NH²-activated biosensor surface can have a specificbinding substance comprising a single-strand DNA capture probeimmobilized on the surface.

The capture probe interacts selectively with its complementary targetbinding partner. The binding partner, in turn, can be designed toinclude a sequence or tag that will bind a “detector” molecule. Adetector molecule can contain, for example, a linker to horseradishperoxidase (HRP) that, when exposed to the correct enzyme, willselectively deposit additional material on the biosensor only where thedetector molecule is present. Such a procedure can add, for example, 300angstroms of detectable biomaterial to the biosensor within a fewminutes.

A “sandwich” approach can also be used to enhance detection sensitivity.In this approach, a large molecular weight molecule can be used toamplify the presence of a low molecular weight molecule. For example, abinding partner with a molecular weight of, for example, about 0.1 kDato about 20 kDa, can be tagged with, for example,succinimidyl6[amethyla(2pyridyldithio) toluamido] hexanoate (SMPT), ordimethylpimelimidate (DMP), histidine, or a biotin molecule.

Detection Apparatus

The photonic crystal biosensors of this disclosure are used inconjunction with an appropriate detection apparatus or instrument. Theparticular nature and construction of the detection apparatus is notespecially important. The detection apparatus detects the luminescentresponse of the fluorophores bound to a sample when the sample andfluorophore are deposited on the surface of the biosensor and the sensoris illuminated with light at a frequency which overlaps the excitationspectrum of the fluorophore. Examples of appropriate detectors forluminescence include CCD-cameras, photomultiplier tubes, avalanchephotodiodes, photodiodes, hybrid photomultiplier tubes, or arraysthereof. The disclosure of the detection apparatus described in U.S.patent application publications U.S. 2003/0027327; 2002/0127565,2003/0059855 and 2003/0032039, U.S. Pat. Nos. 7,023,544, 7,064,844, andpublished PCT application WO 2007/0179024, the contents of each of whichis hereby incorporated herein by reference. Since the detectionapparatus is described in the literature, a further explanation isomitted here for the sake of brevity. The detection apparatus can bearranged to detect in addition changes in the refractive index due tothe coupling of the sample and fluorophore to the sensor surface andresulting shift in the peak wavelength of reflected light. The incidentbeam may be arranged to illuminate the sensing area or all sensing areason one common platform. Alternatively the beam can be arranged toilluminate only a small subarea of the sensing area to be analyzed andthe beam and/or the platform may be arranged so that they can undergorelative movement in order to scan the sensing area of the platform.Accordingly, the detecting apparatus may be arranged in an appropriateway to acquire the luminescence signal intensities of the entire sensingarea in a single exposure step. Alternatively the detection and/orexcitation means may be arranged in order to scan the sensing areasstepwise.

The detection instrument includes a light generating unit whichilluminates the photonic crystal sensor. The light generating unit maycomprise a laser emitting a coherent laser beam. Other suitable lightsources include discharge lamps or low pressure lamps, e.g. Hg or Xe,where the emitted spectral lines have sufficient coherence length, andlight-emitting diodes (LED). The apparatus may also include opticalelements for directing the laser beam so that it is incident on theplatform at an angle θ, and elements for shaping the plane ofpolarization of the coherent beam, e.g. adapted to transmit linearlypolarized light.

Examples of lasers that may be used are gas lasers, solid state lasers,dye lasers, semiconductor lasers. If necessary, the emission wavelengthcan be doubled by means of nonlinear optical elements. Especiallysuitable lasers are argon ion lasers, krypton ion lasers, argon/kryptonion lasers, and helium/neon lasers which emit at wavelengths between 275and 753 nm. Very suitable are diode lasers or frequency doubled diodelasers of semiconductor material which have small dimensions and lowpower consumption.

Another appropriate type of excitation makes use of VCSEL's (verticalcavity surface emitting lasers) which may individually excite therecognition elements on the platform.

In an embodiment in which the photonic crystal is incorporated onto amicroscope slide, the detection instrument may include a microscope forviewing the slide. The microscope may direct a magnified image of thefield of view onto an imaging device such as a charge coupled devicecamera which then captures and stores images of the field of view.Workstations incorporating microscopes and cameras are described in thepatent literature and therefore a detailed discussion of the features ofsuch as system are omitted for the sake of brevity.

FIG. 12 is block diagram of one possible embodiment of a detectioninstrument 190 for use with photonic crystal sensor 10 featuringenhanced excitation and enhanced extraction. The instrument 190 featuresa modified upright fluorescence microscope 195. The instrument 190includes a HeNe laser 200 which directs light 260 through a neutraldensity filter 202 and a beam expander 204. The expanded beam isdirected through an aperture 206 and a ½ wave plate 208 for polarizationand onto a motorized stage of the microscope 195. The motorized stageincludes a linear stage 212 for travel in the X and Y directions. Anadjustable angular stage 210 is mounted to the linear stage 212 and isused to adjust the beam angle θ. Laser light from the stage is directedvia a mirror 209 upwards to a manual stage 214 of the microscope 195.The photonic crystal sensor 10 (e.g., incorporated into a microscopeslide) is placed on the manual stage 214. The laser excitation light 260is tuned to match the excitation band of a fluorophore present in thesample placed on the sensor 10 and causes the fluorophore to emitfluorescence (shown by line 250).

The combination of components 200, 202, 204, 206, 208, 210 and 212provide for an angle-tunable, beam-expanded excitation laser input. Thelaser beam 260 is expanded in the beam expander 204 to ensure uniformexcitation across the sensor 10 and maximum collimation, and croppedwith an aperture 206 to prevent photobleaching outside the imaging area.Alignment of the laser beam to the sensor 10 is achieved using a set ofmirrors (one which is mounted on a precision linear stage 212),polarization is adjusted with a half-wave plate 208, and attenuated witha continuously variable neutral density filter 202. The beam is thenreflected off a gimbal-mounted mirror 209 and incident upon the sensor10. The gimbal mount is controlled with a high precision motor and isitself mounted on a linear stage 212 that also employs a high-precisionmotorized drive. This linear stage 212 ensures the illumination area onthe sensor 10 remains fixed as the excitation beam angle θ is changed.

A halogen lamp housing 220 provides brightfield illumination for thesample placed on the sensor 10. The lamp housing 200 directs white lightto a mirror cube 218 and the light is directed through the objectivelens 216 of the microscope and onto the sensor 10. Magnified,brightfield images of the sample are captured by a simple CMOS camera226. The CMOS camera 226 allows sample focusing and low-resolution imagecapture.

While the variable-angle laser setup of FIG. 12 serves to maximize theenhanced excitation effect of the device, several other design elementsare provided to optimize the enhanced extraction behavior. Inparticular, since the light-emitters on the device (fluorophores, e.g.quantum dots or organic fluorophores) couple more strongly to theresonant extraction modes than do autofluorescing materials within thedevice itself, attempting to exclude all light except these extractionmodes provides maximum signal-to-noise for the output fluorescence. Inorder to accomplish this, there needs to be spatial, frequency, andpolarization filtering. Spatial filtering is accomplished using a lownumerical-aperture (NA) lens 216. While this constrains the extractionmodes to exist within narrow angles of the normal, it provides goodspatial exclusion while still enabling imaging without scanning optics.(Recall the discussion of FIG. 1 and the strongly directional nature ofthe enhanced extraction effect within small angles of vertical).Upstream magnification (not shown) is employed to overcome theresolution limitations of imaging with a low-NA lens. Frequencyselection is done with a narrow-linewidth bandpass filter (228) thatcoincides with the extraction resonance linewidth. Selecting thepolarization for the resonant light extraction provides furthersignal-to-noise improvements.

The fluorescence microscope 195 employs a dichroic mirror in conjunctionwith the filter 224 that reflects the excitation laser light towards aphotodiode detector 222 for purposes of measuring the photonicdispersion of the sensor 10 under test.

An emission filter 228, as described previously, is used to furtherfilter the incident fluorescence emission. This filtered output is fedto a cooled back-thinned electron-multiplier CCD (BT-EM-CCD) 230. Thiscamera 230 has a very large dynamic range to enable high-enhancementmeasurements, and also has excellent sensitivity for pursuingsingle-molecule fluorescence detection.

While presently preferred embodiments have been described withparticularly, variation from the specifics of the disclosed embodimentsare of course possible without departure from the scope of theinvention. All questions concerning scope are to be determined byreference to the appended claims.

1. A detection instrument for a biosensor, comprising: an optical systemdirecting light onto the biosensor comprising a laser light unit, a beamexpander expanding a beam generated by the laser light unit, and adevice for adjusting polarization of the light generated by the laserlight unit; an angle-tunable mirror receiving beam-expanded laser lightand directing the expanded beam to the biosensor; and an imaging systemreceiving light from biosensor, the imaging system comprising an opticalpath containing an objective lens, a narrow-linewidth bandpass filterand a camera; wherein the objective lens comprises the objective lens ofa microscope and wherein the biosensor is in the form of a photoniccrystal formed in a microscope slide.
 2. The detection instrument ofclaim 1, wherein the angle-tunable mirror is mounted to a motorizedstage.
 3. The detection instrument of claim 1, wherein the wavelength ofthe laser light unit is tuned to match the excitation band of afluorophore present in a sample placed on the biosensor.
 4. Thedetection instrument of claim 3, wherein the frequency of the bandpassfilter coincides with a resonance frequency exhibited by the biosensor.5. The detection instrument of claim 1, wherein the camera comprises aCCD imaging camera.
 6. The detection instrument of claim 1, furthercomprising a variable neutral density filter in the optical systemdirecting light onto the biosensor attenuating the output of the laserlight unit.
 7. A detection instrument for a biosensor, comprising: anoptical system directing light onto the biosensor comprising a laserlight unit, a beam expander expanding a beam generated by the laserlight unit, and a device for adjusting polarization of the lightgenerated by the laser light unit; an angle-tunable mirror receivingbeam-expanded laser light and directing the expanded beam to thebiosensor; an imaging system receiving light from biosensor, the imagingsystem comprising an optical path containing an objective lens, anarrow-linewidth bandpass filter and a camera; a brightfieldillumination source for illuminating the biosensor, and a second cameracapturing magnified brightfield images of the biosensor.
 8. Thedetection instrument of claim 1, further comprising a low numericalaperture lens in the optical system directing light onto the biosensor,wherein the tunable mirror receives light from the low numericalaperture lens.
 9. A detection instrument for a biosensor, comprising: anoptical system directing light onto the biosensor comprising a laserlight unit, a beam expander expanding a beam generated by the laserlight unit, and a device for adjusting polarization of the lightgenerated by the laser light unit; an angle-tunable mirror receivingbeam-expanded laser light and directing the expanded beam to thebiosensor; and an imaging system receiving light from biosensor, theimaging system comprising an optical path containing an objective lens,a narrow-linewidth bandpass filter and a camera; a dichoric mirrorreflecting laser light passing through the biosensor and a photodiodedetector receiving the reflected laser light.
 10. The instrument ofclaim 9, wherein the biosensor in is the form of a microwell platehaving a plurality of wells, the wells having a photonic crystal sensorformed therein.
 11. The instrument of claim 1, wherein the angle-tunablemirror is moved such that the camera captures an image of the biosensorat an angle of minimum transmission.
 12. The instrument of claim 1,wherein the biosensor comprises a photonic crystal and, wherein a samplecontaining a fluorophore is applied to the biosensor, and wherein thelight from the biosensor comprises enhanced fluorescence from thefluorophore, the fluorophore excited by the light from the laser lightunit.
 13. A detection system for a biosensor comprising a photoniccrystal incorporated into a microscope slide, combining in combination amicroscope and the detection instrument of claim
 1. 14. A detectioninstrument for a photonic crystal biosensor exhibiting an enhancedexcitation and extraction modes, comprising: a laser light source; anoptical system directing light from the laser light source to thephotonic crystal biosensor, the biosensor responsively andsimultaneously exhibiting (1) an excitation resonance mode having aspectrum which at least partially overlaps an excitation spectrum of afluorophore bound to the biosensor; and (2) an extraction resonance modehaving a spectrum which at least partially overlaps the emissionspectrum of the fluorophore, and a detection system placed to capturelight from the biosensor in the extraction resonance mode.
 15. Theinstrument of claim 14, wherein the detection system includes a cameragenerating an image of the biosensor.
 16. The detection instrument ofclaim 7, further comprising a low numerical aperture lens in the opticalsystem directing light onto the biosensor, wherein the tunable mirrorreceives light from the low numerical aperture lens.
 17. The detectioninstrument of claim 7 further comprising a dichoric mirror reflectinglaser light passing through the biosensor and a photodiode detectorreceiving the reflected laser light.
 18. The instrument of claim 7,wherein the biosensor in is the form of a microwell plate having aplurality of wells, the wells having a photonic crystal sensor formedtherein.
 19. The instrument of claim 7, wherein the angle-tunable mirroris moved such that the camera captures an image of the biosensor at anangle of minimum transmission.
 20. The detection instrument of claim 7,wherein the wavelength of the laser light unit is tuned to match theexcitation band of a fluorophore present in a sample placed on thebiosensor.
 21. The detection instrument of claim 20, wherein thefrequency of the bandpass filter coincides with a resonance frequencyexhibited by the biosensor.
 22. The instrument of claim 9, wherein thebiosensor in is the form of a microwell plate having a plurality ofwells, the wells having a photonic crystal sensor formed therein. 23.The instrument of claim 9, wherein the biosensor comprises a photoniccrystal and wherein a sample containing a fluorophore is applied to thebiosensor, and wherein the light from the biosensor comprises enhancedfluorescence from the fluorophore, the fluorophore excited by the lightfrom the laser light unit.